Peptic ulcers, once thought to result from stress, or excess acidity, or a reduction of the mucosal defense factors in the stomach, are now in a majority of cases, considered to be the result of bacterial infection by Helicobacter pylori. The mounting evidence to this effect is well documented in Helicobacter pylori in Peptic Ulceration and Gastritis, edited by Barry J. Marshall, Richard W. McCallum and Richard L. Guerraut, Blackwell Scientific Publications, Boston, U.S.A. Pertinent Chapters in this work include Chapter four, The Epidemiology of Helicobacter pylori Infection by D. N. Taylor and M. J. Blaser; Chapter seven, Laboratory Diagnosis and Handling of Helicobacter pylori, by T. U. Westblom; and Chapter twelve, Practical Diagnosis of Helicobacter pylori by B. J. Marshall. The history of the discovery H. pylori and its association with gastro intestinal disease is extensively described in "Marshall's Hunch," The New Yorker magazine, pages 64-72, Sep. 20, 1993 and "The Doctor Who Wouldn't Accept No," Reader's Digest magazine, pages 120-124, October 1993.
The effect of treatment of Helicobacter pylori Infection on long term recurrence of Gastric or Duodenal Ulcer is described by David Y. Graham et al in Annals of Internal Medicine 1992; 116: No 9.
Helicobacter pylori has now been shown to be the causative agent for most instances of chronic gastritis (1). See the bibliography below. And, it is now known that, in the absence of aspirin, non-steroidal anti-inflammatory drugs or hypersecretory states, this bacteria is directly implicated in the production of peptic ulcer diseases such as duodenal and benign gastric ulcers (2). And there is now epidemiological data correlating the presence of the H. pylori with gastric cancer (3). The eradication of H. pylori gastritis by antibiotics has been shown to cure peptic ulcers and prevent recurrence (4,5).
It having been seen that the bacteria H. pylori, is present in endoscopically obtained gastric biopsy specimens from both gastric and duodenal ulcer patients and it being known that the enzyme urease is always associated with that bacteria, the concept of diagnosing the presence of such ulcers by testing biopsy specimens for urease suggested itself. Chemical tests for urease were already known in the art. In one such test a urea-containing broth provides a positive urease reaction (hydrolysis of urea) urea+H.sub.2 O urease.fwdarw.NH.sub.3 +CO.sub.2 as indicated by a change in color of the indicator Bacto phenol red from yellow (pH 6.8) to red to cerise at pH 8.1 or more alkaline due to the production of ammonia and/or ammonium carbonate by the urea-urease reaction. See the Difco Manual, 9th edition, Difco Laboratories, Detroit, Mich., (1953). The urea broth described in the Difco Manual was apparently used by B. J. Marshall in the work described in the Rapid Diagnosis of Campylobacteria Associated with Gastritis, The Lancet, Jun. 22, 1985.
This type of urease test has come into commercial and clinical use. In the United States a commercial test product is marketed under the trademark "Clotest." This product is described in U.S. Pat. No. 4,748,113 issued to Barry J. Marshall, on May 31, 1988.
Inasmuch as this test is based upon the reaction of the urease enzyme with urea it will be apparent to any chemist that this reaction can be used to detect either urea or urease, whichever is present in the material to be tested, by the addition of the other material in a test solution. The course of the reaction can be followed by the use of known dye indicators which change color with the changing pH of the reaction mixture as the reaction proceeds. Therefore, references to the detection of urea in the blood or elsewhere are also pertinent. Such references include Mast U.S. Pat. No. 3,395,082 issued Jul. 30, 1968 for a test for urea in aqueous fluids; Chang U.S. Pat. No. 4,101,382 issued Jul. 18, 1978 for a reagent for the determination of urea in biological fluids; and Modrovich U.S. Pat. No. 4,282,316 issued Aug. 4, 1981 for stabilized enzymatic solutions for determining urea.
The detection of preformed urease for the diagnosis of gastrointestinal disorders by the use of water, urea, a bactericide and a pH indicator, is described in by B. J. Marshall Derwent Publication C-86-141647 (1986). The use of urease to determine urea is also described in Derwent Publication C84-128710 (1985). A non-aqueous analytical unit for urease determination, consisting of urea, a buffer for pH 5 to 7.5 and pH indicator, is described in Derwent Publication C90-068529 (1990).
The test composition marketed under the trademark "Clotest" as disclosed in Marshall U.S. Pat. No. 4,748,113 has the following composition:
______________________________________ a) Urea 10-40 g/liter b) A bactericide 1-5 g/liter c) A dye indicator having effective amount A pKa of from about 6.5- 8.5- d) Water To make Wherein the composition 1.0 liter has a pH of from about 5.0-6.5 and in which the pH is at least about one pH unit lower than the pKa of the indicator. ______________________________________
The composition of the Marshall patent uses phenol red alone as the dye indicator.
The method of the Marshall patent is initiated by obtaining a sample of gastric material from the patient by endoscopic biopsy according to procedures well known in the art. The biopsy specimen is then contacted with the test composition set forth above. The test composition is said to be used in liquid form as a rule but may also be gelled. The change in color of the phenol red from yellow at the initial pH (about 5.0-6.5) to red at a pH of about 6.8-9 by the formation of ammonia and/or ammonium carbonate in the hydrolysis reaction, indicates the presence of urease and the Helicobacter pylori bacteria to be identified in the biopsy specimen.
While the test described by Marshall has proven to be highly useful, it has been recognized that it could be improved in certain respects. In the Marshal urease test gastric mucosal biopsy containing H. pylori is placed in solution or an agar gel containing urea, an indicator phenol red, and buffers. The urease in H. pylori converts the urea to ammonia which raises the pH and turns the agar color from a yellow to red, indicating a positive test. According to the package insert in the main commercial phenol red test available (called CLOtest.RTM.), it is recommended that the test be incubated at 30.degree.-40.degree. C. for three hours and that it may take up to 24 hours to develop a positive test. This test relies on the passive diffusion of urease from the cell wall of the bacterium into the agar gel testing solution. Moreover, operating as it does at a pH above 6.5, the test may give a positive result with bacteria other than H. Pylori and thus is not entirely specific for Helicobacter pylori. Specifically, proteus, Pseudomonas and E. Coli species may cause a color change at this level and give a false positive test.
It was apparent therefore that a need has existed in the art for a highly specific test for Helicobacter pylori which would enable diagnosis of a gastrointestinal disorder during a patient's single visit of normal duration to a physician or clinic.